The incidence of infection by Neisseria gonorrhoeae continues to be a global health problem with over 62 million cases of gonorrhea occurring worldwide each year. Our long-term goal is to understand the mechanisms by which the gonococcus, a strict human pathogen, has the ability to infect and cause disease at numerous sites, allowing it to escape the innate host defenses that function at mucosal surfaces. We have evidence that the gonococcus uses efflux pumps to resist a panel of antimicrobial hydrophobic agents (HA) that bathe mucosal surfaces. The action of efflux pumps is also important in the development of gonococcal resistance to antibiotics used previously or presently in the treatment of infections. This proposal is concerned with the action of the MtrC-MtrD-MtrE and FarA-FarB-MtrE efflux pumps, which are responsible for exporting a number of HA that are constitutively present at mucosal surfaces or become available during inflammation. The gonococcal proteins that comprise the core components of the efflux pumps under study have been identified but we have evidence that other proteins function with them during the export process. In order to gain a deeper understanding of how gonococcal efflux pumps function, we will isolate and characterize mutants that express increased or decreased susceptibility to HA that are substrates for each efflux pump (Specific Aim 1). Our previous studies documented that expression of the operons encoding efflux pump proteins are subject to trans and/or cis-acting regulatory control systems that modulate levels of efflux pumps in the cell envelope. The trans-acting elements are a set of DNA-binding that act as transcriptional repressors (MtrR, FarR, MafR and IHF) or activators (MirA), while the cis-acting elements are DNA regulatory sequences that are positioned within or near promoter elements that drive transcription of efflux pump operons. We will define the mechanisms by which these systems control efflux pump gene expression (Specific Aim 2) in order to learn how gonococci modulate production of pump proteins under normal growth conditions or in the presence of sublethal concentrations of HA; the latter situation results in elevated HA-resistance due to enhanced efflux pump gene expression. We have evidence that the MtrR and MtrA regulatory proteins control the expression of genes other than those that encode the core components of the MtrC-MtrD-MtrE efflux pump and that these are important for efflux of HA and virulence. In order to define the repertoire of genes controlled by these DNA-binding proteins and to understand the role of the gene products in gonococcal resistance to antimicrobials, we will use a microarray strategy (Specific Aim 3). The results from the studies in the Specific Aims will advance our knowledge regarding how gonococci and other pathogens resist antimicrobial agents at mucosal surfaces, antibiotics used in the therapy of bacterial diseases, or topical microbicides that may be used in the future to prevent sexually transmitted infections and will increase our understanding of the mechanisms of gonococcal pathogenesis.